The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. We have shown that the protein, GCC185 is needed for tethering of MPRs at the Golgi. To investigate the mechanism of MPR vesicle tethering at the Golgi, we will analyze the structure of this purified Golgi tether using atomic force microscopy. This is important because transport vesicle tethering is a fundamental cell biological process that is poorly understood. We will test if the protein bends on the Golgi, in cells, and whether this bending is needed for its function. We will also determine the consequence of Rab GTPase binding on GCC185's conformation. This is important because GTPase binding is a common feature of Golgins and is likely to reflect the most physiological state of these proteins. We will next add fluorescently labeled, mannose 6-phosphate receptor-containing vesicles and monitor how these engage the GCC185 tether. Where do the vesicles bind? What models best explain how vesicles are tethered at the Golgi? Finally, we will study the very first step in retrograde transport: the loading of Rab9 onto late endosomes. For this, we will determine if Rab9A-specific DENND2 GEFs are part of a late endosomal Rab cascade. Understanding how DENND2 proteins first activate Rab9 will provide key information regarding establishment of the MPR retrograde trafficking pathway on late endosomes and formation of the late endocytic pathway. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.